Generally, recombinant proteins are made as non-natural types when they are expressed in a large scale through the gene manipulation in microbes. In detail, most of the recombinant proteins having a initiator, methionine(MET), in the amino terminus, which were treated inappropriately are produced. The recombinant proteins containing methionine at the amino terminus might induce immunogenic reactions or become being unstable so as not to play an intrinsic role of the protein, in case of being administered to human or other animals. Therefore, it is very important to develop a new method for preparing such a recombinant protein as its natural type protein (See Nature, 1987, 326, 315; J. Bacteriol., 1987, 169, 751–757; Bio/Technology, 1990, 8, 1036–1040; Appl. Microbiol. Biotechnol., 1991, 36, 211–215).
Human growth hormone is a polypeptide hormone which has 22,125 Da of molecular weight, contains a sequence of Phe-Pro-Thr at the amino terminus and is composed of 191 amino acids secreted from human pituitary gland and mostly has been applied to treat pituitary dwarfism (See Raben, M. S., J. Clin. Endocr., 1958, 18, 901). Up to now, human growth hormone has been extracted and purified from pituitary glands of the dead, but is hard to be provided for all the patients due to the limited supply. Recently, several studies regarding expression and separation of the human growth hormone from Escherichia coli, yeast and the like by gene manipulation have been performed. Actually, the human growth hormone produced through the DNA technology has been used for clinical applications (in case of Escherichia coli, See Korean Patent Publication No. 89-1244 and No. 87-701; Korean Patent Laid-open No. 87-2258 and No. 84-8695; in case of yeast, See Korean Patent Publication No. 92-99; Korean Patent Laid-open No. 90-9973 and No. 90-9976).
However, if a human growth hormone is produced by using the recombinant DNA technology described above, one methionine residue, an initiation codon for protein synthesis is bound additionally at the amino terminus except 191 amino acid residues consisting in a natural type human growth hormone and thus a methionyl human growth hormone which is started from the sequence of Met-Phe-Pro-Thr of the amino terminus and composed of 192 amino acid residues is produced. The methionyl human growth hormone has the same biological activities as that of the natural type of human growth hormone (Moore, J. A., Endocrinology, 1988, 122, 2920–2926) and is not reported yet to provoke side effects by presence of the methionine at the amino terminus. However, antibodies might be generated on rare occasions due to the methionine and are reported to be produced in a higher ratio than those from the natural type hormone (Lancet, 1986, Mar. 29, 697).
Hence, there are several approaches to produce a natural type hormone which do not contain the methionine. Concretely, the human growth hormone is produced by one method in which the amino terminus is fused with the carboxy terminus of another protein; and then digested by using a specific protease (See PCT International Application WO 89/12678; European Patent Application EP 20209; European Patent EP 321940); and by another method which comprises (1) expressing growth hormone within cells; (2) digesting methionine while secreted out of host cells; and (3) obtaining a natural type human growth hormone from culture media (See European Patent EP 008832; U.S. Pat. No. 4,755,465; Japanese Patent JP 01273591; European Patent Application EP 306673; Korean Patent Application No. 92-10932). But, there are some disadvantages in these methods illustrated above. Precisely, it is required of complicating construction a new expression vectors and steps of transforming host cells and coincidently to optimize the expression conditions with extra trials.
On the other hand, in case that exogenous protein produced by using recombinant DNA technologies include an extra amino acid at the amino terminus, the aminopeptidase can be exploited to remove the extra amino acid and as a result, the exogenous protein having the same structure with the natural type protein can be yielded easily. Concretely, specific aminopeptidase which cuts selectively only methionine residue present at the amino terminus of the methionyl human growth hormone purified through conventional methods can be used in order to produce a natural type human growth hormone (See PCT International Application WO 86/04609 and WO 86/204527 A1).
Presently, various kinds of aminopeptidases have been demonstrated to prepare a natural type human growth hormone. In detail, the aminopeptidase purified from Aeromonas proteolytica (See PCT International Application WO 86/01229; European Patent EP 0489711, A3, BTG company; Prescott and Wilks, Method in Enzymology, 1976, 44, 530–543), the aminopeptidase purified from porcine kidney (See PCT International Application WO 86/204527 A1; Bio/Technology, 1987, 5, 824–827, Takeda company), the dipeptidyl aminopeptidase purified from Dictyostelium discoidium (See European Patent EP 557076; U.S. Pat. No. 5,126,249, A1, Eli Lilly company), and the aminopeptidase from Streptomyces thermonitripican were reported.
In order to attain the above object, the aminopeptidase should not work on the amino acid sequence of a natural type protein although it removes unnecessary amino acid residues present at the amino terminus of the recombinant protein. Namely, when the original protein has a amino acid sequence starting from X-Y-Z at the amino terminus and the recombinant protein has the amino acid sequence of Met-X-Y-Z-containing an additional methionine at the amino terminus, the aminopeptidase should cut only the methionine residue and not work onto other amino acids (X-Y-Z-) afterward so as to produce the recombinant protein having the same amino acid sequence as that of the natural type protein. Therefore, the aminopeptidase satisfying such an object should be compatible in its substrate specificity depending upon the target protein for industrial applications. Although the enzyme has such a characteristic, it is advantageous to have higher intrinsic activities itself.
Presently, more than several tens of aminopeptidases have been extracted from microbes and the like and disclosed. In common, most enzymes need metal ions such as calcium, zinc or the like in order to be activated. These aminopeptidases have various properties in view of molecular weight, essential metal ions, optimal condition of the reaction, substrate specificity and so on according to microbes, although they have common activities in view of the digestion in amino acid residues at the amino terminus of the protein (See FEMS Microbiol. Rev., 1996, 18, 319–344). Such an aminopeptidase is classified to an exopeptidase and has a property to isolate a amino acid from the amino terminus of substrate protein in orders.
Precisely, these aminopeptidases have been reported and separated, especially from Bacillus sp., for example Bacillus subtilis (See Arch. Biochem. Biophys., 1979, 197, 63–77; Arch. Biochem. Biophys., 202, 540–545, 1980; J. Biochem., 1994, 107, 603–607; Japanese Patent JP 03285684, Diacel-Chem company), Bacillus stearothermophilus (See Meth. Enzymol., 1970, 19, 544–552; Biochem. Biophys. Acta, 1976, 438, 221–220; European Patent EP 101653, Unitika company), Bacillus thuringensis (Biokhimiya, 1984, 49, 1899–1907), Bacillus licheniformis (Arch. Biochem. Biophys., 1978, 186, 383–391; Mikrobiol. Zh., 1989, 51, 49–52) and so on.
In the references mentioned above, the aminopeptidases are purified and analyzed for their enzymatic properties by using leucine-p-nitroanilide, and several dipeptides or the like as a substrate. However, oligopeptides starting from the sequence of Met-X-Pro at the amino termini or proteins starting from Met-X-Pro at the amino termini are not used as a substrate in order to measure enzymatic activities. Therefore, it is not verified yet that these aminopeptidases might be applied to the methionine removal in recombinant proteins containing Met-X-Pro sequence at their amino termini.
Furthermore, the aminopeptidases derived from Bacillus licheniformis have been illustrated as follows. Rodriguez et al. have purified the aminopeptidase derived from Bacillus licheniformis ATCC 12759 and examined its enzymatic property. Also, the inventors of the present invention have disclosed the method for preparing a natural type human growth hormone using the aminopeptidase derived from Bacillus licheniformis, the characterization of the purified aminopeptidase in its enzymatic properties and its amino acid sequence at the amino terminus (Korean Patent Laid-open No. 1998-071239). In this invention, for producing a natural type protein in a large scale through recombinant DNA technologies, the inventors of the present invention have reported that the aminopeptidase could remove only methionine residue in synthetic substrates, oligopeptides, proteins and the like and recognize the specific amino acid sequence of Met-X-Pro (hereinafter, X denotes any amino acid regardless of its kind) and thus be suitable for the production of the natural type human growth hormone (Korean Patent Laid-open No. 1998-071239). These conventional methods have elucidated that the aminopeptidase can be produced from Bacillus licheniformis and exploited to produce a natural type recombinant protein. Besides, they have provided only the information about the partial amino acid sequence of the amino terminus and the total gene of aminopeptidase has not determined yet.
Hence, the inventors of the present invention have tried to obtain novel aminopeptidase for producing a natural type recombinant protein. Concretely, we have cloned a gene of aminopeptidase derived from Bacillus licheniformis, determined its nucleotide sequence and amino acid sequence and expressed the cloned aminopeptidase gene in recombinant bacterial strains. Then, the recombinant protein was identified to have aminopeptidase activities, to be useful for use easily other enzymatic reaction as well as the preparation of natural type proteins. Consequently, the present invention has been completed successfully.